Herpes simplex virus-2 (HSV-2) is one of the most common sexually transmitted infections (STIs) with a prevalence of 45 million in the USA. HSV-2 is transmitted via exposure at the genital mucosal surfaces, leading to the establishment of latency in the sacral ganglia, causing an incurable disease. Although HSV-2- related symptoms can be treated temporarily with antiviral agents, there are no vaccines available for the prevention of this disease. Towards developing an effective HSV-2 vaccine, a clear understanding of the mechanism by which immune responses are generated within the relevant mucosal sites is necessary. Currently, the process of initiation of immune responses within the female genital mucosa is poorly understood. Thus, the goal of the proposed studies is to understand the inductive mechanism of local protective immunity mediated by antigen presenting dendritic cells (DCs) in the female genital tract following infection with HSV-2. In this application, we build on the discoveries made in the first funding period and propose three distinct yet inter-related Aims. In the first Aim, based on our preliminary data demonstrating the requirement for both Toll-like receptor (TLR) and CD4 T help in generating CTL responses following HSV-2 infection, we propose to dissect the mechanisms by which these two signals enable DCs to prime CTL responses against genital herpes infection. Next, based on our demonstration that TLR-mediated recognition of HSV-2 infection by the infected vaginal keratinocytes is necessary for submucosal DCs (SMDCs) to induce robust Th1 immunity in vivo, in the second Aim, we propose to identify the keratinocyte- produced factors responsible for this effect, and to examine their relevance in anti-HSV-2 immunity in vivo. In the final Aim, we propose to examine the in vivo importance of SMDCs in immune priming by generating SMDC inducible-knockout mice. We will create a transgenic mouse line in which the promoter region of the CD301b gene, specifically expressed by the SMDCs, will be used to express diphtheria toxin (DT) receptor- GFP fusion molecule. Upon treatment of such mice with DT, selective depletion of SMDCs will be achieved. This Aim will bring together the knowledge and advances made in Aims 1 & 2 and provide fundamental insights on the interplay between the epithelial cells, SMDCs and other APCs in innate recognition of HSV-2 leading to the generation of adaptive immunity. By providing basic understanding of how vaginal DCs orchestrate the generation of immunity following HSV-2 genital infection, these studies will help to establish critical foundation with which to design immunological interventions and preventative measures against genital herpes and other deleterious STI diseases.